1. Field of the Invention
This invention relates generally to the fields of biochemistry and molecular biology and more specifically to a novel human enzyme, UDP-GlcNAc:Gal.beta.1.fwdarw.3GalNAc (GlcNAc to GalNAc) B1.fwdarw.6 N-acetylglucosaminyltransferase (core 2 B1.fwdarw.6 N-acetylglucosaminyltransferase; C2GnT, EC2.4.1.102), and to a novel acceptor molecule, leukosialin, CD43, for core 2 .beta.1.fwdarw.6 N-acetylglucosaminyltransferase action. The invention additionally relates to DNA sequences encoding core 2 .beta.1.fwdarw.6 N-acetylglucosaminyltransferase and leukosialin, to vectors containing a C2GnT DNA sequence or a leukosialin DNA sequence, to recombinant host cells transformed with such vectors and to a method of transient expression cloning in CHO cells for identifying and isolating DNA sequences encoding specific proteins, using CHO cells expressing a suitable acceptor molecule.
2. Background Information
Most O-glycosidic oligosaccharides in mammalian glycoproteins are linked via N-acetylgalactosamine to the hydroxyl groups of serine or threonine. These O-glycans can be classified into 4 different groups depending on the nature of the core portion of the oligosaccharides (see FIG. 1). Although less well studied than N-glycans, O-glycans likely have important biological functions. Indeed, the presence of O-linked oligosaccharides with the core 2 branch, Gal.beta.1.fwdarw.3(GlcNAc.beta.1.fwdarw.6)GalNAc, has been demonstrated in many biological processes.
Piller et al., J. Biol. Chem 263:15146-15150 (1988) reported that human T-cell activation is associated with the conversion of core 1-based tetrasaccharides to core 2-based hexasaccharides on leukosialin, a major sialoglycoprotein present on human T lymphocytes (see also FIG. 1). A similar increase in hexasaccharides was observed in peripheral blood lymphocytes of patients suffering from T-cell leukemias (Saitoh et al., Blood 77:1491-1499 (1991)), myelogenous leukemias (Brockhausen et al., Cancer Res. 51:1257-1263 (1991)) and immunodeficiency due to AIDS and the Wiskott-Aldrich syndrome (Piller et al., J. Exp. Med. 173:1501-1510 (1991)). In these patients' lymphocytes, changes in the amount of hexasaccharides were caused by increased activity of either UDP-GlcNAc:Gal.beta.1.fwdarw.3GalNAc (GlcNAc to GalNAc) 6-.beta.-D-N-acetylglucosaminyltransferase or core 2 .beta.1.fwdarw.6 N-acetylglucosaminyltransferase (Williams et al., J. Biol. Chem. 255:11253-11261 (1980)). Increased activity of core 2 .beta.1.fwdarw.6 N-acetylglucosaminyltransferase also was observed in metastatic murine tumor cell lines as compared to their parental, non-metastatic counterparts (Yousefi et al., J. Biol. Chem. 266:1772-1782 (1991)).
Increased complexity of the attached oligosaccharides increases the molecular weight of the glycoprotein. For example, leukosialin containing hexasaccharides has a molecular weight of .about.135 kDa, whereas leukosialin containing tetrasaccharides has a molecular weight of .about.105 kDa (Carlsson et al., J. Biol. Chem. 261:12779-12786 and 12787-12795 (1986)).
Fox et al., J. Immunol. 131:762-767 (1983) raised a monoclonal antibody, T305, against human T-lymphocytic leukemia cells. Sportsman et al., J. Immunol. 135:158-164 (1985) reported T305 binding was abolished by neuraminidase treatment, suggesting T305 binds to hexasaccharides. T305 specifically reacts with the high molecular weight form of leukosialin (Saitoh et al., supra, (1991)).
Previous studies indicated poly-N-acetyllactosamine repeats extend almost exclusively from the branch formed by the core 2 .beta.1.fwdarw.6 N-acetylglucosaminyltransferase (Fukuda et al., J. Biol. Chem. 261:12796-12806 (1986)). Consistent with these results, Yousefi et al., supra, (1991) demonstrated that the core 2 enzyme in metastatic tumor cells regulates the level of poly-N-acetyllactosamine synthesis in O-linked oligosaccharides.
Poly-N-acetyllactosamines are subject to a variety of modifications, including the formation of the sialyl Le.sup.x, NeuNAc.alpha.2.fwdarw.3Gal.beta.1.fwdarw.4(Fuc.alpha.1.fwdarw.3)GlcNAc-, or the sialyl Le.sup.a, NeuNAc.alpha.2.fwdarw.3Gal.beta.1.fwdarw.3(Fuc.alpha.1.fwdarw.4)GlcNAc-, determinants (Fukuda, Biochem. Biophys. Acta 780:119-150 (1985)). Such modifications are significant because these determinants, which are present on neutrophils and monocytes, serve as ligands for E- and P-selectin present on endothelial cells and platelets, respectively (see, for example, Larsen et al., Cell 63:467-474 (1990)).
In addition, tumor cells often express a significant amount of sialyl Le.sup.x and/or sialyl Le.sup.a on their cell surfaces. The interaction between E-selectin or P-selectin and these cell surface carbohydrates may play a role in tumor cell adhesion to endothelium during the metastatic process (Walz et al., supra, (1990)). Kojima et al., Biochem. Biophys. Res. Commun. 182:1288-1295 (1992) reported that selectin-dependent tumor cell adhesion to endothelial cells was abolished by blocking O-glycan synthesis. Complex sulfated O-glycans also may serve as ligands for the lymphocyte homing receptor, L-selectin (Imai et al., J. Cell Biol. 113:1213-1221 (1991)).
These reported observations establish core 2 .beta.1.fwdarw.6 N-acetylglucosaminyltransferase as a critical enzyme in O-glycan biosynthesis. The availability of core 2 .beta.1.fwdarw.6 N-acetylglucosaminyltransferase will allow the in vivo and in vitro production of specific glycoproteins having core 2 oligosaccharides and subsequent study of these variant O-glycans on cell-cell interactions. For example, core 2 .beta.1.fwdarw.6 N-acetylglucosaminyltransferase is a useful marker for transformed or cancerous cells. An understanding of the role of core 2 .beta.1.fwdarw.6 N-acetylglucosaminyltransferase in transformed and cancerous cells may elucidate a mechanism for the aberrant cell-cell interactions observed in these cells. In order to understand the control of expression of these oligosaccharides and their function, isolation of a cDNA clone for core 2 .beta.1.fwdarw.6 N-acetylglucosaminyltransferase is a prerequisite. However, the DNA sequence encoding core 2 .beta.1.fwdarw.6 N-acetylglucosaminyltransferase has not yet been reported.
Thus, a need exists for identifying the core 2 .beta.1.fwdarw.6 N-acetylglucosaminyltransferase and the DNA sequences encoding this enzyme. The present invention satisfies this need and provides related advantages as well.